Exhaled Nitric Oxide in Wheezy Infants Predicts Persistent Atopic Asthma and Exacerbations at School Age.

BACKGROUND: There are limited data assessing the predictive value of fraction of exhaled nitric oxide (FENO) in infants/toddlers with recurrent wheezing for asthma at school age. OBJECTIVES: In a cohort of infants/toddlers with recurrent wheezing determine the predictive values of sedated single-breath FENO (SB-FENO) and awake tidal-breathing mixed-expired FENO (tidal-FENO) for active asthma, severe exacerbations, and lung function at age 6 years. METHODS: In 44 infants/toddlers, SB-FENO was measured under sedation at 50 mL/sec in conjunction with forced expiratory flow and volume measurements, and tidal-FENO was measured during awake tidal breathing. Clinical outcomes and lung function were assessed at age 6 years in 36 subjects. RESULTS: Enrollment SB-FENO was significantly higher among subjects with active asthma at age 6 years than among subjects without asthma (36.4 vs. 16.9 ppb, p < 0.0001), and the odds of asthma was 7.6 times greater (OR 7.6; 95% CI 1.8-31.6) for every 10 ppb increase in enrollment SB-FENO. A ROC analysis demonstrated that an enrollment SB-FENO > 31.5 ppb predicted active asthma at age 6 years with an area under the curve (AUC) of 0.92 (95% CI: 0.82-1). SB-FENO was also higher among subjects who experienced severe asthma exacerbations during the year preceding age of 6 years. SB-FENO at enrollment and lung function measures at age 6 years were modestly correlated (FEV1: r = -0.4; FEF25-75: r = -0.41; FEV1/FVC ratio: r=-0.46), and SB-FENO was significantly higher among subjects with bronchodilator responsiveness (BDR) at age 6 years. Tidal-FENO was not predictive of active asthma, exacerbations, or lung function at age 6 years. CONCLUSION: In wheezy infants/toddlers, SB-FENO was predictive of school-age asthma and associated with lung function measures at age 6 years.

Parent- and Child-Reported Asthma Responsibility in School-Age Children: Examining Agreement, Disagreement, and Family Functioning.

INTRODUCTION: The school-age years represent a critical time for children to begin assuming shared asthma management responsibility. This study aimed to describe parent- and child-reported asthma responsibility, examine agreement and disagreement, and explore family functioning as a predictor of agreement/disagreement. METHODS: Twenty children (age range = 6-11 years) and one of their parents participated in this cross-sectional study. Parent-child dyads independently reported on their asthma management responsibility and asthma control. Parents also completed family functioning and demographic questionnaires. RESULTS: There was a significant difference between parent and child asthma responsibility scores (t(19) = 2.46, p < .05), indicating that children saw themselves as assuming greater responsibilities than their parents did. A regression analysis showed that collectively, family functioning predicted 74% of the variance in parent-child disagreement (F(6,15) = 4.17, p < .05). DISCUSSION: Family functioning may be an important factor in promoting shared management of asthma in school-age children.

Fibroblast gene expression following asthmatic bronchial epithelial cell conditioning correlates with epithelial donor lung function and exacerbation history.

Airway remodeling may contribute to decreased lung function in asthmatic children. Bronchial epithelial cells (BECs) may regulate fibroblast expression of extracellular matrix (ECM) constituents and fibroblast-to-myofibroblast transition (FMT). Our objective was to determine if human lung fibroblast (HLF) expression of collagen I (COL1A1), hyaluronan synthase 2 (HAS2), and the FMT marker alpha-smooth muscle actin (α-SMA) by HLFs conditioned by BECs from asthmatic and healthy children correlate with lung function measures and exacerbation history among BEC donors. BECs from asthmatic (n = 23) and healthy children (n = 15) were differentiated at an air-liquid interface (ALI) and then co-cultured with HLFs for 96 hours. Expression of COL1A1, HAS2, and α-SMA by HLFs was determined by quantitative polymerase chain reaction (qPCR). FMT was quantified by measuring HLF cytoskeletal α-SMA by flow cytometry. Pro-collagen Iα1, hyaluronan (HA), and PGE2 were measured in BEC-HLF supernatant. Correlations between lung function measures of BEC donors, and COL1A1, HAS2, and α-SMA gene expression, as well as supernatant concentrations of HA, pro-collagen Iα1, hyaluronan (HA), and PGE2 were assessed. We observed that expression of α-SMA and COL1A1 by HLFs co-cultured with asthmatic BECs was negatively correlated with BEC donor lung function. BEC-HLF supernatant concentrations of pro-collagen Iα1 were negatively correlated, and PGE2 concentrations positively correlated, with asthmatic BEC donor lung function. Expression of HAS2, but not α-SMA or COL1A1, was greater by HLFs co-cultured with asthmatic BECs from donors with a history of severe exacerbations than by HLFs co-cultured with BECs from donors who lacked a history of severe exacerbations. In conclusion, α-SMA and COL1A1 expression by HLFs co-cultured with BECs from asthmatic children were negatively correlated with lung function measures, supporting our hypothesis that epithelial regulation of HLFs and airway deposition of ECM constituents by HLFs contributes to lung function deficits among asthmatic children. Furthermore, epithelial regulation of airway HAS2 may influence the susceptibility of children with asthma to experience severe exacerbations. Finally, epithelial-derived PGE2 is a potential regulator of airway FMT and HLF production of collagen I that should be investigated further in future studies.

Asthmatic bronchial epithelial cells promote the establishment of a Hyaluronan-enriched, leukocyte-adhesive extracellular matrix by lung fibroblasts.

BACKGROUND: Airway inflammation is a hallmark of asthma. Alterations in extracellular matrix (ECM) hyaluronan (HA) content have been shown to modulate the recruitment and retention of inflammatory cells. Bronchial epithelial cells (BECs) regulate the activity of human lung fibroblasts (HLFs); however, their contribution in regulating HLF production of HA in asthma is unknown. In this study, we tested the hypothesis that BECs from asthmatic children promote the generation of a pro-inflammatory, HA-enriched ECM by HLFs, which promotes the retention of leukocytes. METHODS: BECs were obtained from well-characterized asthmatic and healthy children ages 6-18 years. HLFs were co-cultured with BECs for 96 h and samples were harvested for analysis of gene expression, synthesis and accumulation of HA, and subjected to a leukocyte adhesion assay with U937 monocytes. RESULTS: We observed increased expression of HA synthases HAS2 and HAS3 in HLFs co-cultured with asthmatic BECs. Furthermore, we demonstrated greater total accumulation and increased synthesis of HA by HLFs co-cultured with asthmatic BECs compared to healthy BEC/HLF co-cultures. ECM generated by HLFs co-cultured with asthmatic BECs displayed increased HA-dependent adhesion of leukocytes in a separate in vitro binding assay. CONCLUSIONS: Our findings demonstrate that BEC regulation of HA production by HLFs is altered in asthma, which may in turn promote the establishment of a more leukocyte-permissive ECM promoting airway inflammation in this disease.

Fibroblast-myofibroblast transition is differentially regulated by bronchial epithelial cells from asthmatic children.

BACKGROUND: Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs. METHODS: BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin (α-SMA) and flow cytometry was used to assay for α-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments, we investigated the role of TGFß2 in BEC-HLF co-cultures using monoclonal antibody inhibition. RESULTS: Expression of α-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs, but not different than α-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less α-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGFß2 led to similar expression of α-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone. CONCLUSION: These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGFß2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling.

Asthmatic airway epithelial cells differentially regulate fibroblast expression of extracellular matrix components.

BACKGROUND: Airway remodeling might explain lung function decline among asthmatic children. Extracellular matrix (ECM) deposition by human lung fibroblasts (HLFs) is implicated in airway remodeling. Airway epithelial cell (AEC) signaling might regulate HLF ECM expression. OBJECTIVES: We sought to determine whether AECs from asthmatic children differentially regulate HLF expression of ECM constituents. METHODS: Primary AECs were obtained from well-characterized atopic asthmatic (n = 10) and healthy (n = 10) children intubated during anesthesia for an elective surgical procedure. AECs were differentiated at an air-liquid interface for 3 weeks and then cocultured with HLFs from a healthy child for 96 hours. Collagen I (COL1A1), collagen III (COL3A1), hyaluronan synthase (HAS) 2, and fibronectin expression by HLFs and prostaglandin E2 synthase (PGE2S) expression by AECs were assessed by using RT-PCR. TGF-ß1 and TGF-ß2 concentrations in media were measured by using ELISA. RESULTS: COL1A1 and COL3A1 expression by HLFs cocultured with AECs from asthmatic patients was greater than that by HLFs cocultured with AECs from healthy subjects (2.2-fold, P < .02; 10.8-fold, P < .02). HAS2 expression by HLFs cocultured with AECs from asthmatic patients was 2.5-fold higher than that by HLFs cocultured with AECs from healthy subjects (P < .002). Fibronectin expression by HLFs cocultured with AECs from asthmatic patients was significantly greater than that by HLFs alone. TGF-ß2 activity was increased in cocultures of HLFs with AECs from asthmatic patients (P < .05), whereas PGES2 was downregulated in AEC-HLF cocultures (2.2-fold, P < .006). CONCLUSIONS: HLFs cocultured with AECs from asthmatic patients showed differential expression of the ECM constituents COL1A1 and COL3A1 and HAS2 compared with HLFs cocultured with AECs from healthy subjects. These findings support a role for altered ECM production in asthmatic airway remodeling, possibly regulated by unbalanced AEC signaling.